Project Summary/Abstract Despite the critical importance of antiretroviral therapy (ART) adherence in people living with HIV (PLHW), an objective and accurate method to quantify it is still unavailable. While HIV viral load (VL) has been regarded as a surrogate marker of ART adherence, it can lead to inaccurate conclusions in the modern ART era. This is because viremia is a delayed clinical outcome that develops after long-standing non-adherence, and perfect adherence is not required to achieve viral suppression. An emerging pharmacologic method to quantify ART adherence is tenofovir diphosphate (TFV-DP, the phosphorylated anabolite of tenofovir) in dried blood spots (DBS), based on its long intracellular half-life in red blood cells (17 days) with 25-fold accumulation from first dose to steady state. Recent data from the PI (K23AI104315) has demonstrated that TFV-DP in DBS, derived from TDF (TFV-DPTDF), is strongly associated with HIV viral suppression, and that it can predict future viremia in PLWH who are virologically-suppressed. However, the pharmacology of this adherence biomarker remains unknown for TAF-based ART. This is a critical gap given the significant different pharmacology between TDF and TAF, and an indispensable step before it can be implemented in clinical practice. In this revised R01 application, we will advance the field by focusing on TFV-DP in DBS derived from TAF (TFV-DPTAF). Based on our preliminary data, we hypothesize that TFV-DPTAF will be proportional to adherence, and that its variability will be explained by unique patient characteristics in PLWH. We also hypothesize that it will be strongly associated with, and predictive of, viral suppression, and that it will be widely accepted by clinicians as an informative measure of adherence beyond HIV VL. To test these hypotheses, we propose the following aims: Aim 1. Establish the pharmacokinetics (PK) of TFV-DPTAF in DBS in PLWH. This aim will use ingestible biosensors to objectively establish the TFV-DPTAF concentrations associated with actual adherence in PLWH and assess the sources of variability in the drug concentrations. Aim 2. Quantify the relationship between TFV-DPTAF and viral suppression. This aim will estimate the association between TFV-DPTAF and HIV viral suppression (i.e., pharmacodynamics) in a clinical cohort of PLWH on TAF. It will also assess the value of this biomarker as a predictor of future viremia in this cohort. Aim 3. Prospectively evaluate the potential clinical utility of TFV-DPTAF in DBS in PLWH. This aim will assess the perceived utility of this adherence measure by clinicians in the participants from Aim 2, with the goal of understanding how it complements HIV VL in the clinic. Collectively, these studies will advance our understanding on the pharmacology and clinical utility of TFV-DPTAF in PLWH, with the ultimate goal of improving clinical outcomes.